Introduction

HIV-1 was identified in 1983 and subsequently has been shown to be
the etiologic agent of the acquired immunodeficiency syndrome. The
HIV-1 pandemic has grown to become one of the greatest infectious
disease threats to human health and social stability that the world
has ever encountered. An estimated 35 million persons were living
with HIV-1 infection at the beginning of this millennium and more
than 12 million people have already died from HIV-induced disease.
Although effective anti-retroviral therapy has attenuated the
expansion of the epidemic in some industrialized countries, worldwide
there are still an estimated 16,000 new HIV infections occurring
daily. In addition to the vast personal suffering, the loss of young
adult parents, caretakers, and wage-earners, HIV has created an
unprecedented strain on the social and economic infrastructure of
many developing countries, particularly in Sub-Saharan Africa. These
are the facts that make it imperative that the epidemic be controlled
as rapidly as possible through prevention of new infections. Although
education and available public health approaches should be vigorously
pursued, development of a preventive vaccine is the best hope of
controlling the HIV epidemic.

New molecular tools in virology and immunology, new adjuvants, new
gene expression systems, new antigen delivery systems, recent
discoveries in HIV entry and pathogenesis, evidence that natural
immunity is achieved in rare instances, and promising studies of
candidate vaccines in animal

models have provided reasons to hope that developing a safe and
effective AIDS vaccine will be possible. However, some have argued
that preventive vaccination for AIDS will not be possible (1), and
the complex biology of HIV-1 makes this a daunting task.

This review will present current immunologic concepts and
assumptions that form a framework for developing approaches to
vaccine development, relevant data from studies of pathogenesis and
vaccines in humans and animal models, the current status of vaccine
concepts being evaluated in clinical trials, and additional questions
that must be addressed. A more comprehensive review of the issues
involved in HIV vaccine development has been recently published
(2).

Assumptions Relevant to HIV Vaccine Design

Vaccine-induced immunity is possible. While there are many
challenges remaining, the following observations suggest that vaccine
development is achievable:

1) HIV-1 transmission is relatively inefficient

2) transmitted virus may have a restricted set of structural and
genotypic features that are susceptible to immune interference

3) most infections are probably initiated with very few
virions

4) a relatively small impact on transmission efficiency of HIV
between individuals may have a major influence on the epidemic within
a population

5) there are demonstrations of HIV-specific immune responses in
some exposed-but-uninfected persons that suggest it may be possible
for natural immunity to clear virus infection

6) there are examples of vaccine-induced immunity in primate
models of lentivirus infection

9) continued gains in our knowledge of antigen presentation,
cytokines, chemokines, co-stimulatory molecules and their ligands
will lead to new vaccine approaches

10) new vaccine approaches promise to improve the immunogenicity
of vaccine antigens and their delivery into appropriate immunological
compartments.

Timing is everything.

There are several parameters of the vaccine-induced immune
response that will determine its ability to protect the host from
infection or disease including specificity, functional properties,
magnitude, and compartmentalization. However, the most critical
factor is timing. The timing of the immune response with respect to
initial virus infection and spread is particularly important in the
case of HIV-1 infection. One reason for this is that the longer HIV-1
replicates in the host, the more diverse variants evolve that allow
escape from subsequent immune responses. In addition, once HIV-1
resides in the extracellular space of lymph node germinal centers and
in latently infected cellular reservoirs, and is sequestered in the
central nervous system and other sites that are relatively protected
from immune responses, it is unlikely that it can be fully eliminated
from the host. At this stage of infection the immune response to
HIV-1 infection includes a number of potent effector responses that
at best achieve a steady state in which virus clearance matches virus
production. This balance results in a viral load "set point that
correlates with the rate of immune system destruction. The important
advantage of vaccine- induced immune responses is that they are
induced prior to infection and can be recalled more rapidly than
primary effector mechanisms. Therefore the success of vaccination may
hinge upon altering events that occur in the early hours following
HIV-1 exposure.

Vaccines work through induction of adaptive immune responses.
Preventive vaccines work through establishing immunologic memory for
antigenic structures presented by the pathogen or by infected cells.
Therefore, the immunologic "tool box accessible for vaccine-induced
immunity only includes elements of the adaptive immune response. The
basic cellular elements of adaptive immunity include the B and T
lymphocytes. The primary effector mechanisms important for protection
against viruses are antibodies produced by B cells and cytolytic
activity mediated primarily by CD8+ T cells. In addition, there are
soluble factors produced by activated CD4+ and CD8+ T cells that have
anti-viral activity and can influence the differentiation, expansion,
and duration of T cell responses. Elements of the nonadaptive immune
system are important during the initial phases of antigen
presentation and development of the cytokine microenvironment.
However, immunity against subsequent infection will be determined by
adaptive immune responses with memory for key antigens and functional
effector activities that can neutralize the pathogen and rapidly
eliminate infected cells.

Neutralizing antibody and cytolytic T cells are the major
effectors of anti-viral immunity. The correlates of immunity against
HIV-1 have not been defined in an absolute sense, but much is known
about HIV-specific immune responses associated with long-term
survival and maintenance of low viral loads. In addition, there is a
general understanding about how different elements of the adaptive
immune response should work and these concepts can be tested against
observations made in studies of the natural history of HIV infection
in humans or experimental data from animal models (4). Alternative
vaccine-inducible effector mechanisms mediated by the adaptive immune
response may ultimately be shown to have a role in protection (5-11)
but in this review I will focus on classical neutralizing antibody
and CD8+ cytolytic T cell activities. There is often debate and
speculation about which component of the adaptive immune system is
most important for immunity. However, there is abundant evidence for
HIV and other virus infections that both antibody and CD8+ CTL are
important and perform complementary roles in protection from and
control of infection. CD4+ T cells are also of obvious importance,
especially for influencing differentiation patterns and expansion of
selected lymphocyte populations, but their role as a direct effector
of virus clearance in less clear. Therefore, another assumption is
that CD4+ T cells will be induced in the process of achieving the
appropriate antibody and CD8+ CTL responses, and will not be
specifically addressed in this paper.

Antibody is the only component of the adaptive immune response
that can neutralize a virus particle prior to infection of a cell and
is the only immune response associated with protection for any
currently licensed vaccines. Antibody titers can be sustained at high
levels in serum and in mucosal secretions and be present at the time
of infection. This is unlike T cells which only recognize virus in
the context of an already infected cell, and require a few days for
activation and expansion of memory populations to respond. Therefore,
an effective neutralizing antibody response will be a critical
component of vaccine induced immunity, because it can prevent
infection and thereby reduce inoculum size and establishment of
latently infected cells.

Neutralization is defined as the ability to reduce infectivity of
cell-free virus usually measured in susceptible cells in culture.
While this aspect of antibody activity is thought to be the key
function associated with protection from infection. There is some
debate about the mechanism of neutralization. There are reports of
specific neutralizing epitopes suggesting the site of antibody
binding is important (12-16). Another view is that neutralization
occurs when a threshold level of the virion surface is covered by
antibody that binds the native envelope oligomer regardless of
specificity (17). In either case, it is clear that T cell
line-adapted viruses are more susceptible to neutralization than
primary field isolates which poses a major hurdle for achieving this
immunologic endpoint (18, 19).

T cells recognize virus infected cells by specific interactions
between the T cell receptor and 8- 10 amino acid peptides processed
from viral antigens and presented in the context of major
histocompatibility complex (MHC) molecules. Therefore, T cells can
only clear virus effectively after infection hasoccurred.The
recognition is restricted by the MHC molecule, which means that the
particular epitopes recognized by a given individual will depend on
the set of inherited alleles encoding the MHC molecules. While each
person should have the capacity to recognize multiple epitopes among
the antigens included in HIV-1, the hierarchy of recognition or
epitope dominance may vary even among individuals who share MHC
haplotypes. These issues suggest that the epitope repertoire in a
vaccine will need to have enough breadth to encompass all the
relevant MHC haplotypes of potential vaccinees. In addition, it will
be important to induce a broad response in each individual against
several viral antigens to diminish the possibility of immune escape
through genetic variation and to allow for host selection of dominant
epitopes. The need for CD4+ T lymphocytes to initiate the adaptive
immune response presents a dilemma since these cells are the major
target for HIV-1 infection. The problem is how to effectively induce
protective immunity against HIV-1 without putting vaccine-induced
HIV-specific CD4+ T cells at risk of infection. This emphasizes the
need for effective immune responses, preexistent at the time of HIV
exposure, so that virus clearance can be accomplished before the
burden of infected cells is sufficient to maintain persistent
infection. While CD4+ T cells may have some capacity for lysis of
HIV-infected cells (20) and production of anti-viral cytokines, the
major role is in shaping the immune response by establishing a
microenvironment with a particular cytokine composition. For HIV and
most other viruses, induction of a Type 1 cytokine profile
(production of IL-12, IL-2, and IFN- ) is more likely to provide
protection than induction of Type 2 cytokines (IL-4, IL-5, IL-13).
Initial priming with vectors and the use of adjuvants other than alum
(which promotes Type 2 responses) would provide an advantage in this
regard.

CD8+ T cells are the principal effector mechanism of the adaptive
immune response to clear virus infected cells. This has been
demonstrated exhaustively in murine models of LCMV, Sendai virus,
influenza virus, respiratory syncytial virus, ectromelia, herpes
simplex, and others (21-27). The CD8+ lymphocyte recognizes a
virus-infected cell through a cognate interaction between the T cell
receptor and a processed peptide epitope presented in the grove of a
MHC class I molecule. The lysis of the infected cells occurs through
the production and secretion of perforin and granzymes that penetrate
the target cell membrane and induce apoptosis. FasL is also
upregulated on the activated CD8+ T cell which can bind Fas on the
target cell and induces apoptosis through other pathways. CD8+ T
cells also produce cytokines with anti-viral properties like IFN- and
TNF- , in addition to other soluble factors that may play a role in
virus inhibition. The T cell response causes cytopathology not only
of the virus-infected cell but to a varying degree in bystander
cells. This again points to the importance of clearing virus rapidly
to diminish the overall cytopathology and illness associated with the
immune response to infection.

Acceptable definitions of vaccine-induced immunity. The outcome of
infection in the setting of vaccine-induced immunity could range from
complete prevention of infection to immune-mediated enhancement of
disease. None of the currently licensed vaccines for other viral
pathogens are known to fully prevent infection, and most are
effective because they limit the replication and spread of the
pathogen below the threshold for clinical expression of disease.
Ideally, a vaccine against HIV-1 will either prevent infection or
result in a transient infection that is rapidly cleared before the
establishment of latently infected cells or widespread dissemination.
Vaccine-induced immune responses may not be sufficient to prevent
persistent infection. However, if low virus loads could be maintained
to protect the individual from disease and to limit transmission to
others, this would also be an acceptable outcome for vaccine-induced
immunity.

Data from Studies in Humans and Animal Models

Antibody can prevent HIV infection. It has been directly proven
using passive antibody studies in nonhuman primate models of
lentivirus infection that sufficient levels of neutralizing antibody
can prevent infection. Studies evaluating polyclonal anti-HIV-1
antiserum (28) or monoclonal anti-V3 antibody in HIV-1 infected
chimpanzees (29) or polyclonal serum in SIV-infected macaques (30)
have shown that when sufficiently high antibody titers are present
prior to intravenous challenge that lentivirus infection can be
prevented. Importantly, antibody mediated protection has also been
demonstrated against SHIV with an envelope glycoprotein derived from
a dual tropic primary HIV isolate, and the protection could be
correlated with in vitro neutralizing activity (31). More recently,
passive prophylaxis using HIV immune globulin combined with two
monoclonal antibodies has protected macaques from vaginal challenge
with SHIV (32), and a mixture of three neutralizing monoclonal IgG1
antibodies given to pregnant macaques has protected their infants
from SHIV oral challenge (33). Definitive evidence of
antibody-mediated protection in studies of active immunization has
been more difficult to demonstrate, but there is an example from
early studies performed with whole inactivated SIV vaccines that is
provocative. In these studies it was shown that antibodies to cell
constituents incorporated into virions during production of challenge
stocks were the best correlate of protection (34-37). When the virus
used to produce vaccine was grown in human cells, and the virus
challenge stock was grown in the same human cells, allogeneic
responses to the human proteins incorporated by the virus were the
dominant mechanism of protection (34,38,39). Studies done with
vaccine produced in monkey cells did not show consistent protection.
Even though the antibody response was not specific for virus-encoded
antigens, this represents an example of vaccine-induced
antibody-mediated protection suggesting that protection through
induction of virus-specific antibodies may be achievable. When SIV
immune globulin was given one day after intravenous challenge with
SIV, infection was not prevented, but disease progression was delayed
in some animals (40). This again illustrates that the timing of
immune responses are critical to the outcome of infection and that
preexisting immunity gives the host a distinct advantage.

T cells can control HIV infection.

Control of the initial viremia associated with primary HIV
infection temporally correlates with the appearance of CD8+ cytotoxic
T lymphocytes (41,42), and mutations in specific CTL epitopes can be
detected in the residual virus population (43-47). In addition,
HIV-specificCD8+ CTL activity has been demonstrated in a small subset
of uninfected, seronegative commercial sex workers in The Gambia and
in Kenya suggesting transient infection may have occurred inducing
protective immunity mediated by CD8+ CTL (48,49). In persons who
remain uninfected despite significant occupational exposure to HIV-1
contaminated material, studies have also focused on HIV-specific T
cell responses. Although HIV-specific antibodies can not be detected,
PBMCs show lymphoproliferative activity when stimulated with
HIV-specific peptides (50). HIV-specific CTL responses have also been
seen in this cohort (51), suggesting that transient infection may
have occurred and been cleared with natural immune defenses. Another
subset of persons infected with HIV-1 have persistent infection, but
do not progress to AIDS for greater than 12 years. Some of these
individuals are infected with virus isolates that replicate poorly
(52,53). However, others are infected with viruses that have normal
replication capacity, but have maintained a strong and broad set of
humoral and cellular HIV-specific immune responses that appears to be
responsible for their delayed disease progression. This has been
associated with HIV-specific CD4+ T cell proliferation (54) and
strong CD8+ CTL activity against multiple epitopes (55,56). Another
clue to the importance of T cell responses in the control of HIV has
come from the evaluation of HIV- infected persons treated with highly
active anti-retroviral therapy (HAART) soon after primary infection.
When these persons undergo structured treatment interruptions there
is a transient rise in the virus load which results in a boost of
functional T cell activity and subsequent control of virus load
without HAART (57).

The most compelling evidence for the importance of CD8+ CTL for
controlling lentivirus infection comes from studies of pathogenesis
and vaccine evaluation in nonhuman primate models. The CD8+ CTL
response is the best correlate of viremia control after primary SIV
infection in macaques, similar to the findings in HIV-infected humans
as discussed above (58). There are now several studies using nucleic
acid or other recombinant vector approaches that have demonstrated
induction of CD8+ CTL responses with a weak or absent antibody
response, does not protect from lentivirus infection, but reduces
viral load and delays disease progression. One of the early
demonstrations of this was in macaques immunized with recombinant MVA
(modified vaccinia Ankara) prior to challenge of macaques with SIV.
Vaccination did not prevent infection, and the CTL cell response was
associated with delayed disease progression (59). Subsequent studies
have shown similar patterns (60-67). As approaches are taken to
optimize the CD8+ CTL response, such as the addition of an IL-2
adjuvant to a recombinant DNA vaccine regimen, nearly complete
control of subsequent SHIV infection can be achieved (67). These data
are consistent with the premise that vaccines able to establish a
preexisting expanded population of HIV-specific CD8+ CTL are likely
to delay disease progression in HIV-infected persons.

Clinical Trials of Candidate HIV Vaccines

Overview of concepts evaluated. Clinical trials in seronegative
volunteers have been performed to evaluate the safety and
immunogenicity of candidate AIDS vaccines in more than 3500 subjects.
Several recombinant envelope products, rgp120 or rgp160, produced in
insect, yeast or mammalian cells formulated with a variety of
adjuvants have been evaluated in clinical trials. Peptides tested to
date have been derived from envelope V3 loop or gag sequences of
clade B or multiple clades. They have been presented conjugated to an
oligolysine backbone, as a lipopeptide conjugate, mixed with
adjuvant, or as a fusion protein with the self-assembling yeast
protein Ty as a particle. They have been administered intramuscularly
in the deltoid or anterior thigh (to target lymph nodes that also
drain the rectal mucosa), rectally and orally as Ty-gag virus-like
particles, and orally encapsulated in polylactide co-polymers.Live
recombinant vectors including vaccinia, canarypox, and salmonella
have been evaluated as well as nucleic acid based vaccines. These
vectors have been delivered by a variety of routes and have been
constructed to express both single or multiple HIV-1 antigens. In
addition, there have been studies evaluating schedule of
administration and combination approaches using more than one product
in the immunization regimen. These studies are listed in Table 1 and
referenced when possible. This review will summarize the findings of
the studies to date without including the details of each individual
product. Because there have been no significant safety concerns other
than unacceptable local reactogenicity associated with a few selected
adjuvants, I will focus on vaccine immunogenicity particular the
ability to induce neutralizing antibody and CD8+ CTL
responses.Vaccine-induced antibody responses in clinical trials.
Neutralizing antibody responses have been induced by immunization
with recombinant envelope glycoproteins alone or in combination with
poxvirus vectors. The antibody response to immunization with rgp120
alone is in general maximal after the third or fourth injection, is
dose dependent, and can be attenuated unless there is a several month
interval between injections. Serum antibody titers have a relatively
short half-life, and while they can be boosted the titers generally
achieve their peak level after the third or fourth injections.
Repeated boosting does not prolong the half-life significantly.
Therefore, it is likely that recombinant envelope glycoprotein
products may find their greatest utility in boosting antibody
responses in subjects primed with recombinant vector vaccines (99),
or other strategies that can induce MHC class I-restricted CTL
responses. This combination approach not only adds the CD8+ CTL
component to the immune response, but results in a more durable
antibody response. The initial recombinant envelope glycoprotein
products were derived from sequences of syncytium-inducing, T cell
line-adapted (TCLA), CXCR-4 utilizing X4 viruses from clade B. Newer
products, such as the VaxGen B/B product incorporate sequences from
primary isolates which utilize CCR5 (R5) combining the rgp120 from
HIV-1MN and the rgp120 from HIV-1GNE8 (110). Phase I and II studies
have defined how parameters of dose, schedule, and formulation affect
immunogenicity of purified protein subunit preparations as primary
immunogens and as booster immunogens given in combination with other
vaccine modalities. The principal findings related to vaccine-induced
antibody responses in clinical trials of candidate HIV vaccines
include:

1) While type-specific neutralization can be induced, particularly
to the vaccine antigen, neutralization of typical primary R5 HIV
isolates is not induced (111). There are some reports of
neutralization of selected R5 HIV strains, but these are strains that
are easier to neutralize in general, and how this will translate into
protection against more typical neutralization resistant strains is
not known.

2) Antibody is induced that can bind oligomeric, R5 virus
presented on the surface of virus-infected cells (112). This suggests
that the monomeric envelope products currently being tested can
produce antibody that recognizes oligomeric envelope structures, even
though the affinity and specificity is not sufficient to result in
virus neutralization.

3) Antigens produced in mammalian cells induce higher titer of
neutralizing antibody against TCLA virus than those produced in
baculovirus or yeast systems (69,73,77,78).

4) Recombinant gp120 products induce less binding antibody, but
more neutralizing antibody than rgp160 products (71,77,78). As noted
above, the neutralizing activity does not include primary isolate R5
viruses.

5) A four dose immunization regimen using envelope glycoprotein is
more effective for antibody induction when there is a several month
interval between doses (77,78). Intervals of at least 3 to 4 months
between the second, third, and fourth immunization increase the
magnitude of response.

6) A rapid (every month) vaccination schedule using envelope
glycoproteins alone results in attenuation of antibody responses
after the fourth dose (77). Titers of both binding and neutralizing
antibody activities are reduced after a monthly immunization schedule
using rgp120 in MF59 (77). The attenuating effect of rapid dosing is
not as apparent with other adjuvants (72).

7) A fifth dose of rgp120, regardless of interval, does not boost
antibody response, but only returns it to previous level.

8) The half-life of vaccine antigen-specific antibody titers is 3
months in subjects receiving only rgp120 envelope glycoprotein,
regardless of number of doses. The half-life is extended with gp160
antigens, and is also more prolonged when rgp120 immunization is
preceded by priming with poxvirus vectors. The factors underlying
antibody maintenance have not been defined.

9) Priming with one subtype and boosting with another demonstrates
subtype-specificity in antibody response (102). When a subject is
initially immunized with rgp120 derived from a clade B, TCLA X4 HIV
strain, subsequent boosting with another clade B strain does not
broaden the response significantly, and does not boost the response
to the new envelope antigen as well as to the original rgp120.

10) Antigen dose effects on magnitude of antibody production are
dependent on the adjuvant formulation. QS21 appears to allow a
reduction in the antigen dose by more than 10-100 fold without
affecting the magnitude of antibody response (113).

11) Peptide vaccines in general have been weakly immunogenic with
one exception. This was a complex peptide that contains T helper
epitopes from the C4 domain of gp120 and neutralizing antibody and
CTL epitopes from the V3 domain. Peptides from four strains of HIV-1
were combined in incomplete Freund's adjuvant and administered
intramuscularly. Neutralizing activity against HIV-1MN was detected
in 75 of subjects after the second dose. However, the study was
terminated prematurely because of the development of sterile
abscesses in a few vaccinees.

12) Recombinant gp160 vaccinia immunization induces an antibody
response to the HIV envelope that is slow in developing, often not
being detectable until 100 days after inoculation (100),

14) Specificity of the antibody response in subject primed with
recombinant vaccinia then boosted with recombinant envelop
glycoprotein is determined more by the initial antigen expressed by
the recombinant vector than by the subsequent envelope antigen given
as booster (101,102),

16) In subjects immunized with recombinant HIV-1LAI gp160 vaccinia
and boosted with HIV- 1LAI gp160 produced in baculovirus and
formulated with alum, the dominant antibody response in vaccinees was
directed against a gp41 epitope (aa 720-740) that was not a major
target for antibodies produced by HIV-1LAI-infected persons
(114).

In summary, neutralizing antibody responses against TCLA viruses
induced by the most immunogenic formulations are still 5-10 fold
lower than those produced by HIV-1 infection. The responses are
type-specific with a relatively short half-life, and are unable to
neutralize typical primary isolate R5 viruses.

Vaccine-induced CD8+ CTL responses in clinical trials. Induction
of HIV-specific CD8+ CTL responses requires the delivery of vaccine
antigens into the cytoplasmic compartment of an antigen presenting
cell (APC) for display in a MHC class I molecule on the cell surface.
Therefore, vector-based approaches or nucleic acid vaccines that rely
on antigen production within the target cell are most effective.
Delivering vaccine antigens as purified proteins or even whole
inactivated virus will primarily access the endocytic pathway for
antigen presentation and lead to CD4+ T cell activation. While this
is critical for antibody production and important for supporting CD8+
CTL development, it is not sufficient for inducing CD8+ CTL. In some
cases a novel adjuvant or delivery system is able to provide access
for these types of vaccines into the cytoplasmic compartment, but in
general vector-based vaccines, including nucleic acids, are more
potent methods for inducing CD8+ CTL. One exception is the use of
peptides which incorporate a T cell epitope that can bind directly to
an MHC class I molecule on the cell surface and induce CD8+ CTL
responses. Vector-based vaccines, beginning with recombinant vaccinia
products, were first evaluated in clinical trials in the late 1980`s
with the expressed purpose of achieving vaccine-induced CD8+ CTL
responses. The induction of CD8+ CTL responses has been a primary
focus of clinical trials since the mid 1990's. The principal findings
related to vaccine-induced CD8+ CTL responses in clinical trials of
candidate HIV vaccines include:

2) HIV-specific CD8+ CTL can be detected in a majority of subjects
receiving recombinant poxvirus vectors, and in a subset CTL activity
is detectable for 18 months. The activity is at the threshold of
detection in classical 51Cr release assays requiring in vitro
stimulation and is only detected in 15-30 of subjects at any given
time point. However, unlike antibody responses, vaccine-induced CTL
responses are broadly reactive (117). CTL induced by recombinant
canarypox vectors has been shown to lyse target cells infected with
primary R5 HIV-1 isolates from multiple clades (117). CD8+ CTL
effectors have also been isolated from rectal mucosa from vaccinees
suggesting that T cells induced by parenteral vaccination may provide
some level of protection at mucosal surfaces (McElrath et al.,
unpublished observations). Not only is classical MHC class
I-restricted cytolytic activity induced, but vaccine-induced
noncytolytic CD8+-mediated suppression of HIV-1 replication (118) has
been demonstrated in recipients of recombinant canarypox vaccines
(119).

3) Envelope subunits can induce CD4+ CTL, but rarely induce CD8+
CTL even when formulated with novel adjuvants (77,120). This is an
expected result of obligate processing through the endocytic pathway
leading to MHC class II presentation. New adjuvants and delivery
systems may improve MHC class I presentation of purified protein
antigens, but it is unlikely to ever approach the efficiency of
antigens produced intracellularly by approaches such as nucleic acid
immunization, live recombinant vectors, or live attenuated
vaccines.

4) In one study with the Ty-gag-VLP without alum CD8+ CTL
responses were detected in a few subjects (84).

In summary, vaccine approaches that are currently being evaluated
in clinical trials can induce HIV-specific CD8+ CTL activity that is
durable and can lyse cells infected with typical primary R5 HIV-1
isolates from multiple clades.

Future Directions

Timeline for HIV vaccine development. Although the need for an
effective vaccine against HIV is urgent, what is a realistic timeline
for identifying the approaches and products needed to achieve
vaccine-induced immunity? In a May 18, 1997 commencement address at
Morgan State University President Clinton challenged the scientific
community to have a vaccine within 10 years. Thus, the vaccine
development effort is motivated by political as well as humanitarian,
social, ethical, and scientific purposes. To meet this timeline, I
believe the following goals will have to be achieved:

2) development of new approaches to optimize the breadth and
magnitude of vaccine-induced memory CTL

3) performance of a large scale trial to test the concept that a
vaccine-induced CD8+ CTL response will modify infection rates or
alter the course of naturally acquired HIV infection.

Induction of R5 neutralizing antibody activity. Many R5 viruses
are difficult to neutralize even with high titer, type-specific
serum. The basis for the relative neutralization resistance is an
active area of current research. Empiric approaches are being pursued
to find envelope glycoprotein structures that can induce broad
neutralizing antibody and include:

1) triggering the envelope glycoprotein into an intermediate
structure present during the membrane fusion process

4) expression of envelope glycoproteins in vectors and virus-like
particles

5) multi-epitope combinations. In addition, a more systematic
approach combining information from structural biology, molecular
biology, and epitope mapping to produce novel envelope structures is
being pursued. Solving the problem of how to produce an antigenic
structure that can induce a neutralizing antibody response effective
against typical R5 HIV-1 isolates represents an important step toward
addressing additional questions about the antibody response
including:

1) will local induction of mucosal antibody be necessary for
protection from HIV infection or will high titer serum antibody be
sufficient

2) can broadly neutralizing antibody be induced with a single
antigenic structure, or will mixtures of envelope glycoprotein
structures be required

3) can durable antibody responses be induced, or will repeated
booster immunizations be required?

Duration, magnitude, kinetics, and breadth of CTL response. There
is evidence from studies in both humans and animal models that a
robust CD8+ CTL response can control HIV infection. However, the
character of those responses and relative degree of protection for a
given level and breadth of CTL response have not been defined. There
are candidate vaccines currently in Phase II clinical trials that can
induce CD8+ CTL responses in a portion of subjects. Whether this
level of CTL is sufficient for control of HIV infection is not known.
However, based on studies in nonhuman primates, it is likely that
vaccine induction of a robust CD8+ CTL response will lower the viral
load set point. The large number of vector based vaccine delivery
approaches, emergence of cytokine adjuvants to specifically direct
selected immune responses, and advances in methods used to enumerate
T cell responses promise that strategies for consistent
vaccine-induction of high magnitude CTL responses are within reach in
a time frame of about 5 years. Future studies may need to address the
following issues:

1) how broad does the CTL response need to be to control viremia,
in terms of number and dominance hierarchy of epitopes

2) does the CTL response measured in peripheral blood accurately
predict control of viremia, or will specific induction of CTL
responses in mucosal tissue or lymph nodes be required

3) can the kinetics of the CTL response or efficiency of killing
be influenced by vaccine formulation and delivery to improve
protection, or is the outcome only determined by the magnitude of the
response, and

The importance of efficacy trial evaluation. One of the next major
steps in vaccine development will be the performance of larger scale
trials in higher risk populations. The appropriate timing, vaccine
approach, trial design, and trial location for such a study are
issues of current controversy and debate. The performance of a Phase
III clinical trial should be based on:

1) its potential for defining a biological impact of the vaccine
on HIV-induced disease based on results from animal model studies and
Phase I/II trials

2) its potential for answering questions about correlates of
immunity

3) the importance of establishing a benchmark against which future
vaccine design and development can be measured.

For example, if a Phase III study can be designed and executed
that will show whether CD8+ CTL can control HIV viremia, the current
vaccine strategies with potential CTL-inducing capacity would be
accelerated with a focus on optimizing the magnitude of CD8+
induction. If induction of CD8+ CTL is not associated with any level
of protection, then issues of breadth and compartmentalization of
responses will need to be addressed more rigorously in animal
models.

The ultimate vaccine that can prevent persistent HIV-1 infection
will probably require a conceptual breakthrough in the understanding
of how to elicit broadly neutralizing antibody against primary R5
HIV-1 isolates, and will also involve a number of iterative steps to
achieve optimal HIV specific CD8+ CTL responses. However, a vaccine
aimed at control of viremia, delayed disease progression, and reduced
transmission, based on induction of HIV-specific CD8+ CTL could have
a significant impact on the AIDS epidemic, and may be within our
grasp using currently available technology.

Acknowledgments

I thank John Mascola, Gary Nabel, and Peter Wright for reviewing
the manuscript. The work was supported in part by UO1-AI-47985